Sterically hindered poly(ethylene glycol) alkanoic acids and derivatives thereof

ABSTRACT

The invention provides a sterically hindered polymers and conjugates formed therefrom that comprise a water-soluble and non-peptidic polymer backbone having at least one terminus covalently bonded to an alkanoic acid or alkanoic acid derivative prior to conjugation, wherein the carbon adjacent to the carbonyl group of the acid or acid derivative group has an alkyl or aryl group pendent thereto. The steric effects of the alkyl or aryl group allow greater control of the hydrolytic stability of polymer derivatives. The polymer backbone may be poly(ethylene glycol).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/689,979, filed on Jan. 19, 2010, now U.S. Pat. No. 7,960,502, whichis a continuation of U.S. application Ser. No. 12/215,086, filed on Jun.25, 2008, now U.S. Pat. No. 7,674,879, which is a continuation of U.S.application Ser. No. 11/716,202, filed on Mar. 9, 2007, now U.S. Pat.No. 7,405,266, which is a continuation of U.S. application Ser. No.11/264,546, filed on Nov. 1, 2005, now U.S. Pat. No. 7,205,380, which isa continuation of U.S. application Ser. No. 10/813,601, filed on Mar.30, 2004, now U.S. Pat. No. 6,992,168, which is a continuation of U.S.application Ser. No. 10/283,890, filed on Oct. 30, 2002, now U.S. Pat.No. 6,737,505, which is a divisional of U.S. application Ser. No.09/741,933, filed on Dec. 20, 2000, now U.S. Pat. No. 6,495,659, whichclaims the benefit of U.S. Provisional Application No. 60/171,784, filedon Dec. 22, 1999, all of which are herein incorporated by reference intheir entireties.

FIELD OF THE INVENTION

This invention generally relates to water-soluble and non-peptidicpolymers, and methods of controlling the hydrolytic properties of suchpolymers.

BACKGROUND OF THE INVENTION

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, also known as poly(ethylene oxide), abbreviated PEO, tomolecules and surfaces is of considerable utility in biotechnology andmedicine. In its most common form, PEG is a linear polymer terminated ateach end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol represents the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—where n typically ranges from about 3 to about 4000.

PEG is commonly used as methoxy-PEG-OH, or mPEG in brief, in which oneterminus is the relatively inert methoxy group, while the other terminusis a hydroxyl group that is subject to ready chemical modification. Thestructure of mPEG is given below.CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

Random or block copolymers of ethylene oxide and propylene oxide, shownbelow, are closely related to PEG in their chemistry, and they can besubstituted for PEG in many of its applications.HO—CH₂CHRO(CH₂CHRO)_(n)CH₂CHR—OHwherein each R is independently H or CH₃.

To couple PEG to a molecule, such as a protein, it is often necessary to“activate” the PEG by preparing a derivative of the PEG having afunctional group at a terminus thereof. The functional group is chosenbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For example, the functional group could be chosento react with an amino group on a protein in order to form a PEG-proteinconjugate.

PEG is a polymer having the properties of solubility in water and inmany organic solvents, lack of toxicity, and lack of immunogenicity. Oneuse of PEG is to covalently attach the polymer to insoluble molecules tomake the resulting PEG-molecule “conjugate” soluble. For example, it hasbeen shown that the water-insoluble drug paclitaxel, when coupled toPEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336(1995).

The prodrug approach, in which drugs are released by degradation of morecomplex molecules (prodrugs) under physiological conditions, is apowerful component of drug delivery. Prodrugs can, for example, beformed by bonding PEG to drugs using linkages which are degradable underphysiological conditions. The lifetime of PEG prodrugs in vivo dependsupon the type of functional group linking PEG to the drug. In general,ester linkages, formed by reaction of PEG carboxylic acids or activatedPEG carboxylic acids with alcohol groups on the drug, hydrolyze underphysiological conditions to release the drug, while amide and carbamatelinkages, formed from amine groups on the drug, are stable and do nothydrolyze to release the free drug.

Use of certain activated esters of PEG, such as N-hydroxylsuccinimideesters, can be problematic because these esters are so reactive thathydrolysis of the ester takes place almost immediately in aqueoussolution. It has been shown that hydrolytic delivery of drugs from PEGesters can be favorably controlled to a certain extent by controllingthe number of linking methylene groups in a spacer between the terminalPEG oxygen and the carbonyl group of the attached carboxylic acid orcarboxylic acid derivative. For example, Harris et al., in U.S. Pat. No.5,672,662, describe PEG butanoic acid and PEG propanoic acid (shownbelow), and activated derivatives thereof, as alternatives tocarboxymethyl PEG (also shown below) when less hydrolytic reactivity inthe corresponding ester derivatives is desirable.PEG-OCH₂CH₂CH₂CO₂HPEG butanoic acidPEG-O—CH₂CH₂CO₂HPEG propanoic acidPEG-O—CH₂CO₂Hcarboxymethyl PEG

In aqueous buffers, hydrolysis of esters of these modified PEG acids canbe controlled in a useful way by varying the number of —CH₂— spacersbetween the carboxyl group and the PEG oxygen.

There remains a need in the art for further methods of controlling thehydrolytic degradation of activated polymer derivatives.

SUMMARY OF THE INVENTION

The invention provides a group of water-soluble and non-peptidicpolymers having at least one terminal carboxylic acid or carboxylic acidderivative group. The acid or acid derivative group of the polymer issterically hindered by the presence of an alkyl or aryl group on thecarbon adjacent to the carbonyl group of the carboxylic acid (α-carbon).The steric effect of the alkyl or aryl group enables greater control ofthe rate of hydrolytic degradation of polymer derivatives. For example,both activated carboxylic acid derivatives, such as succinimidyl esters,and biologically active polymer conjugates resulting from the couplingof the polymers of the invention to biologically active agents, such assmall drug molecules, enzymes or proteins, are more hydrolyticallystable due to the presence of the α-carbon alkyl or aryl group.

The sterically hindered polymers of the invention comprise awater-soluble and non-peptidic polymer backbone having at least oneterminus, the terminus being covalently bonded to the structure

wherein:

L is the point of bonding to the terminus of the polymer backbone;

Q is O or S;

m is 0 to about 20;

Z is selected from the group consisting of alkyl, substituted alkyl,aryl and substituted aryl; and

X is a leaving group.

Examples of suitable water-soluble and non-peptidic polymer backbonesinclude poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide),poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene,polyoxazoline, poly(N-acryloylmorpholine), and copolymers, terpolymers,and mixtures thereof. In one embodiment, the polymer backbone ispoly(ethylene glycol) having an average molecular weight from about 200Da to about 100,000 Da.

Examples of the Z moiety include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, t-butyl, and benzyl. In one embodiment, Zis a C₁-C₈ alkyl or substituted alkyl.

The leaving group, X, can be, for example, halogen, such as chlorine orbromine, N-succinimidyloxy, sulfo-N-succinimidyloxy,1-benzotriazolyloxy, hydroxyl, 1-imidazolyl, and p-nitrophenyloxy.

The invention also includes biologically active conjugates of thepolymers of the invention and biologically active agents and methods ofmaking such conjugates.

By changing the length or size of the alkyl or aryl group used as the Zmoiety, the polymers of the invention offer an increased ability tocontrol and manipulate the hydrolytic stability of polymer derivativesprepared using the polymers. Better control of the rate of hydrolyticdegradation enables the practitioner to tailor polymer constructs forspecific end uses that require certain degradation properties.

DETAILED DESCRIPTION OF THE INVENTION

The terms “functional group”, “active moiety”, “activating group”,“reactive site”, “chemically reactive group” and “chemically reactivemoiety” are used in the art and herein to refer to distinct, definableportions or units of a molecule. The terms are somewhat synonymous inthe chemical arts and are used herein to indicate that the portions ofmolecules that perform some function or activity and are reactive withother molecules. The term “active,” when used in conjunction withfunctional groups, is intended to include those functional groups thatreact readily with electrophilic or nucleophilic groups on othermolecules, in contrast to those groups that require strong catalysts orhighly impractical reaction conditions in order to react. For example,as would be understood in the art, the term “active ester” would includethose esters that react readily with nucleophilic groups such as amines.Typically, an active ester will react with an amine in aqueous medium ina matter of minutes, whereas certain esters, such as methyl or ethylesters, require a strong catalyst in order to react with a nucleophilic

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pHs, e.g., under physiological conditions for anextended period of time, perhaps even indefinitely. Hydrolyticallyunstable or degradable linkages means that the linkages are degradablein water or in aqueous solutions, including for example, blood.Enzymatically unstable or degradable linkages means that the linkage canbe degraded by one or more enzymes. As understood in the art, PEG andrelated polymers may include degradable linkages in the polymer backboneor in the linker group between the polymer backbone and one or more ofthe terminal functional groups of the polymer molecule. For example,ester linkages formed by the reaction of PEG carboxylic acids oractivated PEG carboxylic acids with alcohol groups on a biologicallyactive agent generally hydrolyze under physiological conditions torelease the agent. Other hydrolytically degradable linkages includecarbonate linkages; imine linkages resulted from reaction of an amineand an aldehyde (see, e.g., Ouchi et al., Polymer Preprints, 38(1):582-3(1997), which is incorporated herein by reference.); phosphate esterlinkages formed by reacting an alcohol with a phosphate group; hydrozonelinkages which are reaction product of a hydrazide and an aldehyde;acetal linkages that are the reaction product of an aldehyde and analcohol; orthoester linkages that are the reaction product of a formateand an alcohol; peptide linkages formed by an amine group, e.g., at anend of a polymer such as PEG, and a carboxyl group of a peptide; andoligonucleotide linkages formed by a phosphoramidite group, e.g., at theend of a polymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalorganism, including but not limited to viruses, bacteria, fungi, plants,animals, and humans. In particular, as used herein, biologically activemolecules include any substance intended for diagnosis, cure mitigation,treatment, or prevention of disease in humans or other animals, or tootherwise enhance physical or mental well-being of humans or animals.Examples of biologically active molecules include, but are not limitedto, peptides, proteins, enzymes, small molecule drugs, dyes, lipids,nucleosides, oligonucleotides, cells, viruses, liposomes, microparticlesand micelles. Classes of biologically active agents that are suitablefor use with the invention include, but are not limited to, antibiotics,fungicides, anti-viral agents, anti-inflammatory agents, anti-tumoragents, cardiovascular agents, anti-anxiety agents, hormones, growthfactors, steroidal agents, and the like.

The terms “alkyl,” “alkene,” and “alkoxy” include straight chain andbranched alkyl, alkene, and alkoxy, respectively. The term “lower alkyl”refers to C1-C6 alkyl. The term “alkoxy” refers to oxygen substitutedalkyl, for example, of the formulas —OR or —ROR¹, wherein R and R¹ areeach independently selected alkyl. The terms “substituted alkyl” and“substituted alkene” refer to alkyl and alkene, respectively,substituted with one or more non-interfering substituents, such as butnot limited to, C3-C6 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and thelike; acetylene; cyano; alkoxy, e.g., methoxy, ethoxy, and the like;lower alkanoyloxy, e.g., acetoxy; hydroxy; carboxyl; amino; loweralkylamino, e.g., methylamino; ketone; halo, e.g. chloro or bromo;phenyl; substituted phenyl, and the like. The term “halogen” includesfluorine, chlorine, iodine and bromine.

“Aryl” means one or more aromatic rings, each of 5 or 6 carbon atoms.Multiple aryl rings may be fused, as in naphthyl or unfused, as inbiphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Substituted aryl” is aryl having one or more non-interfering groups assubstituents.

“Non-interfering substituents” are those groups that yield stablecompounds. Suitable non-interfering substituents or radicals include,but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₁-C₁₀ alkoxy, C₇-C₁₂ aralkyl, C₇-C₁₂ alkaryl, C₃-C₁₀cycloalkyl, C₃-C₁₀ cycloalkenyl, phenyl, substituted phenyl, toluoyl,xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₇-C₁₂ alkoxyaryl, C₇-C₁₂aryloxyalkyl, C₆-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NRC(O)—(C₁-C₁₀ alkyl),—C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ thioalkyl, —C(O)O—(C₁-C₁₀ alkyl), —OH,—SO₂, ═S, —COOH, —NR₂, carbonyl, —C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃,—C(O)NR₂, —(C₁-C₁₀ alkyl)-S—(C₆-C₁₂ aryl), —C(O)—(C₆-C₁₂ aryl),—(CH₂)_(m)—O—(CH₂)_(n)—O—(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂, —NRC(S)NR₂, salts thereof, andthe like. Each R as used herein is H, alkyl or substituted alkyl, arylor substituted aryl, aralkyl, or alkaryl.

The invention provides a sterically hindered polymer, comprising awater-soluble and non-peptidic polymer backbone having at least oneterminus, the terminus being covalently bonded to the structure

wherein:

L is the point of bonding to the terminus of the polymer backbone;

Q is O or S;

m is 0 to about 20;

Z is selected from the group consisting of alkyl, substituted alkyl,aryl and substituted aryl; and

X is a leaving group.

The polymer backbone of the water-soluble and non-peptidic polymer canbe poly(ethylene glycol) (i.e. PEG). However, it should be understoodthat other related polymers are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to be inclusive and not exclusive in this respect. The termPEG includes poly(ethylene glycol) in any of its forms, including alkoxyPEG, difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendentPEG, or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 3 to about 2000, is one usefulpolymer in the practice of the invention. PEG having a molecular weightof from about 200 Da to about 100,000 Da are particularly useful as thepolymer backbone.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH)_(m) in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble and non-peptidic polymer backbonesis by no means exhaustive and is merely illustrative, and that allpolymeric materials having the qualities described above arecontemplated.

Examples of suitable alkyl and aryl groups for the Z moiety includemethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,t-butyl, and benzyl. In one embodiment, Z is a C₁-C₈ alkyl orsubstituted alkyl.

The optional CH₂ spacer between the α-carbon and the Q moiety canprovide additional dampening effect on the rate of hydrolyticdegradation of the molecule. In one embodiment, m is 1 to about 10.

The X moiety is a leaving group, meaning that it can be displaced byreaction of a nucleophile with the molecule containing X. In some cases,as when X is hydroxy, the group must be activated by reaction with amolecule such as N,N′-dicyclohexylcarbodiimide (DCC) in order to make itan effective leaving group. Examples of suitable X moieties includehalogen, such as chlorine and bromine, N-succinimidyloxy,sulfo-N-succinimidyloxy, 1-benzotriazolyloxy, hydroxyl, 1-imidazolyl,and p-nitrophenyloxy. In one aspect, the polymer has a terminalcarboxylic acid group (i.e. X is hydroxyl).

In one embodiment, the polymer of the invention has the structure

wherein:

POLY is a water-soluble and non-peptidic polymer backbone, such as PEG;

R′ is a capping group; and

Q, m, Z and X are as defined above.

R′ can be any suitable capping group known in the art for polymers ofthis type. For example, R′ can be a relatively inert capping group, suchas an alkoxy group (e.g. methoxy).

Alternatively, R′ can be a functional group. Examples of suitablefunctional groups include hydroxyl, protected hydroxyl, active ester,such as N-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, protected amine,hydrazide, protected hydrazide, thiol, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, and tresylate. The functionalgroup is typically chosen for attachment to a functional group on abiologically active agent. As would be understood, the selected R′moiety should be compatible with the X group so that reaction with Xdoes not occur.

As would be understood in the art, the term “protected” refers to thepresence of a protecting group or moiety that prevents reaction of thechemically reactive functional group under certain reaction conditions.The protecting group will vary depending on the type of chemicallyreactive group being protected. For example, if the chemically reactivegroup is an amine or a hydrazide, the protecting group can be selectedfrom the group of tert-butyloxycarbonyl (t-Boc) and9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is athiol, the protecting group can be orthopyridyldisulfide. If thechemically reactive group is a carboxylic acid, such as butanoic orpropionic acid, or a hydroxyl group, the protecting group can be benzylor an alkyl group such as methyl or ethyl. Other protecting groups knownin the art may also be used in the invention.

Specific examples of terminal functional groups in the literatureinclude N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zaplipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPoly(ethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zaplipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al. Macrolol. Chem.180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11:141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461). In addition, two molecules of the polymer ofthis invention can also be linked to the amino acid lysine to form adi-substituted lysine, which can then be further activated withN-hydroxysuccinimide to form an active N-succinimidyl moiety (see, e.g.,U.S. Pat. No. 5,932,462). All of the above references are incorporatedherein by reference.

R′ can also have the structure —W-D, wherein W is a linker and D is abiologically active agent. Alternatively, the polymer structure can be ahomobifunctional molecule such that R′ is -Q(CH₂)_(n)CHZC(O)X, whereinQ, m, Z and X are as defined above.

An example of a multi-arm polymer of the invention is shown below:

wherein:

POLY is a water-soluble and non-peptidic polymer backbone, such as PEG;

R is a central core molecule, such as glycerol or pentaerythritol;

q is an integer from 2 to about 300; and

Q, m, Z and X are as defined above.

Further examples of the polymers of the invention include polymers ofthe structure

wherein:

PEG is poly(ethylene glycol); and

X, m and Z are as defined above.

The polymers of the invention, whether activated or not, can be purifiedfrom the reaction mixture. One method of purification involvesprecipitation from a solvent in which the polymers are essentiallyinsoluble while the reactants are soluble. Suitable solvents includeethyl ether or isopropanol. Alternatively, the polymers may be purifiedusing ion exchange, size exclusion, silica gel, or reverse phasechromatography.

In all the above embodiments, the presence of the α-alkyl or α-arylgroup (Z) confers upon the polymer greater stability to hydrolysis dueto the steric and electronic effect of the alkyl or aryl group. Thesteric effect may be increased by increasing the size of the alkyl oraryl group, as would be the case in replacing methyl with ethyl. Inother words, as the number of carbon atoms in Z increases, the rate ofhydrolysis decreases. As noted above, use of this steric effect may alsobe applied in combination with the electronic effect obtained byvariation in the distance of the Q moiety from the carboxyl group (i.e.control of the value of m). By controlling both m and Z, the rate ofhydrolysis can be regulated in a more flexible manner.

Since the enzyme catalyzed reactions that cause enzymatic degradationinvolve exact spatial fits between the enzyme active site and thepolymer, steric effects can be very important in these reactions aswell. The polymers of the invention can also be used to better regulateor control enzymatic degradation in addition to hydrolytic degradation.

When coupled to biologically active agents, the polymers of theinvention will help regulate the rate of hydrolytic degradation of theresulting polymer conjugate. As an example, when the polymers of theinvention are coupled with alcohols or thiols to form esters orthioesters respectively, the esters or thioesters are more stable tohydrolysis. Thus, a drug bearing an alcohol or thiol group may bederivatized with a polymer of the invention and the hydrolytic releaseof the drug from such esters or thiolesters can be controlled by choiceof the α-alkyl or α-aryl group.

The invention provides a biologically active polymer conjugatecomprising a water-soluble and non-peptidic polymer backbone having atleast one terminus, the terminus being covalently bonded to thestructure

wherein:

L is the point of bonding to the terminus of the polymer backbone;

Q is O or S;

m is 0 to about 20;

Z is selected from the group consisting of alkyl, substituted alkyl,aryl and substituted aryl;

W is a linker; and

D is a biologically active agent.

The linker W is the residue of the functional group used to attach thebiologically active agent to the polymer backbone. In one embodiment, Wis O, S, or NH.

Examples of suitable biologically active agents include peptides,proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides,oligonucleotides, cells, viruses, liposomes, microparticles andmicelles.

The invention also includes a method of preparing biologically activeconjugates of the polymers of the invention by reacting a polymer ofFormula I with a biologically active agent.

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention.

EXPERIMENTAL Example 1 Preparation of mPEG-O—CH₂CH(CH₃)CO₂H andmPEG-O—CH₂CH(CH₃)CO₂NS (NS═N-succinimidyl)

Reactions:

1. Preparation of mPEG₅₀₀₀-O—CH₂CH(CH₃)CN

MPEG₅₀₀₀₀H (4.0) g) and methacrylonitrile (1.0 ml) were stirred forthree days at room temperature in a mixture of benzene (5.0 ml),dichloromethane (6.5 ml), and KOH (50% in H₂O; 0.15 ml). To theresulting mixture was added 200 ml of 10% aqueous NaH₂PO₄. The mixturewas stirred for 10 minutes before extracting with 200 ml ofdichloromethane (100+50+50 ml). The organic phase was dried over MgSO₄,concentrated, and precipitated into ethyl ether (50 ml). The precipitatewas collected by filtration and dried under vacuum at room temperatureto obtain 3.17 g of white powder. NMR: (dmso-d6, ppm): 1.0438 (d,α-CH₃); 2.55 (m, CH); 3.51 (br m, PEG-CH₂CH₂—O—).

2. Preparation of mPEG₅₀₀₀-O—CH₂CH(CH₃)CONH₂

mPEG₅₀₀₀-O—CH₂CH(CH₃)CN (3.17 g) was dissolved in 14 ml of concentratedHCl and the solution was stirred three days at room temperature. Theresulting solution was diluted to 300 ml with water and 45 g of NaCl wasadded. The product was extracted with dichloromethane (3×100 ml) and theextract dried over MgSO₄. The solution was concentrated and the productprecipitated in ethyl ether (50 ml). The product was collected byfiltration and dried under vacuum at room temperature to obtain 2.6 g ofwhite powder. NMR (dmso-d6, ppm): 0.714 (d, α-CH₃); 3.51 (br m, PEG—CH₂CH₂—O—).

3. Preparation of mPEG₅₀₀₀-O—CH₂CH(CH₃)CO₂H

A solution of 2.6 g of mPEG₅₀₀₀-O—CH₂CH(CH₃)CONH₂ in 100 ml of 8% KOHwas stirred at room temperature for three days and the pH was thenadjusted to 2.0 with HCl. The product was extracted with 100 ml ofmethylene chloride and the extract dried over MgSO₄. The solution wasthen concentrated and the product precipitated by addition to 200 ml ofethyl ether. The product was collected by filtration and dried undervacuum at room temperature to obtain 1.7 g of white powder. The productwas further purified by chromatography on DEAE sepharose with the columnfirst eluted with water and then with 1 M NaCl. The product wasextracted from the NaCl eluent with methylene chloride and the organiclayer dried over MgSO₄. The methylene chloride solution was concentratedand the product precipitated from about 30 ml of ethyl ether. It wascollected by filtration, and dried under vacuum at room temperature toobtain 0.8 g of white powder. Gel permeation chromatography onUltrahydrogel 250 displayed a single peak.

¹H NMR (dmso-d6, ppm): 1.035 (d, α-CH₃); 2.55 (m, CH); 3.51 (br m, PEGbackbone CH₂). The integral ratio of the PEG backbone protons to that ofthe alpha methyl protons indicated 100% substitution.

4. Preparation of CH₃—O-PEG₅₀₀₀-O—CH₂CH(CH₃)CO₂NS (NS═N-succinimidyl)

CH₃—O-PEG₅₀₀₀-O—CH₂CH(CH₃)CO₂H (0.6 g) was dissolved in 50 ml ofmethylene chloride, N-hydroxysuccinimide (0.0144 g) andN,N-dicyclohexylcarbodiimide (0.026) in 2 ml of methylene chloride wasadded. After stirring overnight, the mixture was filtered and thefiltrate concentrated under vacuum. The product was precipitated byaddition of the filtrate to isopropanol, then collected by filtrationand dried under vacuum to yield 0.4 g of white powder. Comparison ofintegration of the PEG backbone protons with those on the NS groupindicated 100% substitution.

¹H NMR (ppm, dmso-d6): 1.20 (d, CH ₃—CH); 2.81 (s, NS); 3.51 (br m, PEG—CH₂CH₂—O—).

Example 2 Preparation of mPEG-O—CH₂CH₂CH(CH₃)CO₂H andmPEG-O—CH₂CH₂CH(CH₃)CO₂NS

Reactions:

1. Preparation of CH₃—O-PEG-O—CH₂CH₂C(CH₃)(CO₂H)₂

Diethyl methylmalonate (9.6 ml) in 150 ml of dry dioxane was addeddropwise to NaH (2.4 g) in 60 ml of toluene under argon. MPEG₅₀₀₀mesylate (30 g) in 250 ml of toluene was azeotropically distilled toremove 150 ml of toluene and the residue was added to the above diethylmethylmalonate solution. After refluxing the mixture for 3-4 hours, itwas evaporated under vacuum to dryness and dried in vacuo overnight. Thedried material was then dissolved in 200 ml of 1N NaOH, the solution wasstirred for 2 days at room temperature, and the pH adjusted to 3 with 1NHCl. NaCl was added to the solution to a concentration of about 15% andthe mixture was then extracted with 350 ml of CH₂Cl₂ in severalportions. The combined extracts were dried over Na₂SO₄, concentratedunder vacuum and the product precipitated by addition ofisopropanol/ether (1:1). The product was collected by filtration anddried under vacuum overnight to obtain 24.7 g of product as a whitepowder. GPC (Ultrahydrogel 250) showed the product to be 98% pure.

¹H NMR (dmso-d6, ppm): 1.27 (s, CH₃—C); 1.96 (t, CH₂CH ₂—C); 3.51 (br m,PEG —CH₂CH₂—O—).

2. Preparation of CH₃—O-PEG₅₀₀₀-O—CH₂CH₂CH(CH₃)CO₂H

CH₃—O-PEG₅₀₀₀-O—CH₂CH₂C(CH₃)(CO₂H)₂ (20 g) was dissolved in 300 ml oftoluene and the resulting solution was refluxed for 3 hours. Thesolution was then concentrated under vacuum and the residue precipitatedwith isopropanol/ether (1:1), collected by filtration, and dried undervacuum overnight to obtain 18.8 g of white powder. GPC (Ultrahydrogel250) indicated the product to be 95% pure.

¹H NMR (dmso-d6, ppm): 1.061 (d, CH ₃—CH); 2.40 (q, CH); 1.51 (m, CH₂—CH); 1.80 (m, CH ₂—CH₂—CH); 3.51 (br m, PEG —CH₂CH₂—O—).

3. Preparation of CH₃—O-PEG₅₀₀₀-O—CH₂CH₂CH(CH₃)CO₂NS(NS═N-succinimidyl)

CH₃—O-PEG₅₀₀₀-O—CH₂CH₂CH(CH₃)CO₂H (3.8 g) was dissolved in 40 ml ofmethylene chloride and N-hydroxysuccinimide (0.094 g, 1.07 equiv.) andN,N-dicyclohexylcarbodiimide (0.166 g, 1.07 equiv.) in 3 ml of methylenechloride was added. After stirring overnight, the mixture was filteredand the filtrate concentrated under vacuum. The product was precipitatedby addition of the filtrate to a 1:1 mixture of isopropanol and ethylether then collected by filtration and dried under vacuum to yield 3.2 gof white powder. Comparison of integration of the PEG backbone protonswith those on the NS group indicated >95% substitution.

¹H NMR (ppm, dmso-d6): 1.235 (d, CH ₃CH—); 1.76 (m, 1.90 m, —O—CH₂ CH₂CH—); 2.81 (s, CH₂CH₂ on NS;) 2.91 (m, —O—CH₂CH₂CH—); 3.51 (br m, PEG—CH₂CH₂—O—).

Example 3 PEGylation of Lysozyme with Activated α-Alkylalkanoic Acids

To 4 ml of lysozyme solution (3 mg/ml) in 50 pH 6.5 buffer (50 mM sodiumphosphate/50 mM NaCl) was added 20 mg of the N-succinimidyl ester of thePEG alkanoate and the progress of the reaction at 22° C. was monitoredby capillary electrophoresis at a wavelength of 205 nm. The area of thepeak corresponding to unreacted protein was plotted against time and thehalf-life of the lysozyme in the PEGylation reaction was determined fromthat plot. The half-life using N-succinimidyl mPEG_(5K)α-methylpropanoate was 100 minutes, while that of N-succinimidylmPEG_(5K) α-methylbutanoate was 120 minutes. The half-life forPEGylation using either of the non-α-alkylated analogues, mPEG_(5K)N-succinimidyl propanoate or mPEG_(5K) N-succinimidyl butanoate, was 30minutes.

Example 4 Hydrolysis Rates of N-Succinimidyl mPEG α-Alkylalkanoates

Hydrolysis studies were conducted at pH 8.1 and 25° C. In a typicalexperiment, 1-2 mg of the N-succinimidyl ester of the PEG alkanoate orPEG α-alkylalkanoate were dissolved in 3 ml of buffer and transferred toa cuvette. The absorbance at 260 nm was monitored using a MolecularDevices SpectraMax Plus uv-visible spectrophotometer. The hydrolytichalf-life was determined from the first-order kinetic plot. ForN-succinimidyl mPEG_(5K) α-methylpropanoate and N-succinimidyl mPEG_(5K)α-methylbutanoate, the half-lives for hydrolysis were 33 minutes and 44minutes respectively, while for the corresponding non-alkylatedanalogue, N-succinimidyl mPEG_(5K) propanoate and mPEG_(5K) butanoate,the half-life was 20 minutes.

Example 5 8-arm-PEG_(20KDa) α-Quinidine α-methylbutanoate

8-arm-PEG_(20KDa) α-methyl butanoic acid (2.0 g, 0.1 mmol) wasazeotropically dried in vacuo with CHCl₃ (3×50 ml) and was redissolvedin CH₂Cl₂ (25.0 ml). To this clear solution was added quinidine (0.50 g,1.5 mmol), DMAP (0.15 g, 1.2 mmol), and HOBt (cat.). DCC (0.310 g, 1.5mmol in 1 ml of CH₂Cl₂) was then added and the mixture was allowed tostir at room temperature under argon for 17 h. The mixture was thenconcentrated in vacuo and the residual syrup was dissolved in toluene(100 ml) and filtered through a plug of Celite. The toluene was removedin vacuo at 45° C. and the residue was treated with 5 ml of CH₂Cl₂ andtriturated with 2-propanol (300 ml). Further drying in vacuo afforded apure product (2.0 g, 99%) with 100% substitution as indicated by ¹H NMR.

Example 6 Hydrolysis study of 8-arm-PEG_(20KDa)-Quinidineα-methylbutanoate by reverse phase HPLC

A C-18 column (Betasil C18, 100×2, 5 Keystone Scientific) was used in aHP-1100 HPLC system. Eluent A was 0.1% TFA in water, while eluent B wasacetonitrile.

For the hydrolysis study in pure buffer, the quinidine conjugate wasdissolved in 10 mM phosphate buffer for a final concentration of 8mg/ml. The resulting solution was pipetted into sealed vials (0.2 mleach) at 37° C. At timed intervals, a vial was taken and to it was added0.2 ml of acetonitrile. After filtration, the sample was analyzed byRP-HPLC with UV detector at wavelength of 228 nm. Least squares kinetictreatment of the data yielded a half-life of 46 hours for hydrolysis.

Example 7 (Pivaloyloxy)methyl mPEG_(5KDa)-α-methylbutanoate

mPEG_(5KDa) α-methylbutanoic acid (16.8 g, 3.4 mmol) was dissolved inacetonitrile (500 ml) and was concentrated in vacuo to about 100 ml.Dichloromethane (100 ml) was added under argon and the solution wasallowed to stir at room temperature. To this clear, colorless solutionwas added DBU (2.4 mL, 16.2 mmol) followed by chloromethyl pivalate (2.4ml, 16.6 mmol). The solution was allowed to stir at room temperatureunder argon for 17 h. The solution was then concentrated to dryness,dissolved in 2-propanol (300 ml), and cooled in an ice bath to give awhite solid that was collected by filtration. Further drying in vacuogave (pivaloyloxy)methyl mPEG_(5KDa)-α-methylbutanoate (14.5 g, ˜86%) asa white solid. ¹H NMR (dmso-d₆, 300 MHz) δ 1.08 (d, 3H, J=7.1 Hz,OCH₂CH₂CH(CH₃)COPOM), 1.14 (s, 9H, OCH₂CO(CH₃)₃), 1.55-1.69 (m, 2.8H,OCH₂CH_(A)H_(B)CH(CH₃)COPOM), 1.73-1.85 (m, 1.3H,OCH₂CH_(A)H_(B)CH(CH₃)COPOM), 2.49-2.60 (m, OCH₂CH₂CH—(CH₃)COPOM), 3.51(bs, 454H, PEG backbone), 5.70 (s, 1.9H, COCH₂POM)(POM=pivaloyloxymethy).

1. A method of preparing a conjugate, wherein the method comprises reacting a sterically hindered polymer having the structure:

wherein: R is a central core molecule; POLY is a water-soluble and non-peptidic polymer backbone; Q is O or S; m is 1 to about 20; Z is selected from the group consisting of alkyl, substituted alkyl, aryl and substituted aryl; and X is a leaving group; and q is an integer from 2 to about 300; with a biologically active agent comprising a nucleophilic functional group suitable to displace X under conditions suitable to form a conjugate.
 2. The method of claim 1, wherein R is glycerol residue.
 3. The method of claim 1, wherein R is a pentaerythritol residue.
 4. The method of claim 1, wherein q is
 8. 5. The method of claim 1, wherein POLY is poly(ethylene glycol).
 6. The method of claim 5, wherein the poly(ethylene glycol) has an average molecular weight from about 200 Da to about 100,000 Da.
 7. The method of claim 1, wherein Z is a C₁-C₈ alkyl or substituted alkyl.
 8. The method of claim 1, wherein Z is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and benzyl.
 9. The method of claim 1, wherein m is 1 to about
 10. 10. The method of claim 1, wherein X is selected from the group consisting of chlorine, bromine, hydroxyl, N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotriazolyloxy, 1-imidazolyl, and p-ntirophenyloxy. 